Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSECs) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a proinflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear. Methods: Primary mouse LSECs isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH resulting from a choline-deficient high-fat diet (CDHFD) or FFC diet were also treated with two structurally distinct GSK3 inhibitors (LY2090314 and elraglusib [9-ING-41]). Results: Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phosphoproteome identified glycogen synthase kinase (GSK)-3β as the major kinase hub in MASH. GSK3β-activating phosphorylation was increased in primary human LSECs treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC- and CDHFD-fed mice, respectively. Immunophenotyping using cytometry by mass cytometry by time of flight of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells. Conclusion: GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and could serve as a potential therapeutic strategy for treating human MASH. Impact and Implications: LSECs under lipotoxic stress in MASH develop a proinflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identified GSK3 as the major driver of LSEC endotheliopathy, examined its pathogenic role in myeloid cell-associated liver inflammation, and defined the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for the future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver disease and MASH, as well as those interested in drug development.

Maintaining pH is critical for proper cellular and organ function. To measure intracellular pH, we made a novel genetically encoded protein, pHire, that increases red fluorescence with increasing pH. Thus, pHire allows for the coupling of multiple fluorescent tags without spectral overlap. We have expressed pHire in mammalian epithelial cells, Drosophila, and mice. In this study, our goal was to characterize pHire’s spectral properties by expression in human cells (HEK293) and in E.coli. To biophysically characterize pHire, we made a maltose binding protein (MBP)-pHire fusion protein to purify from E.coli. Pelleting individual colony growths revealed pink bacterial pellets which fluoresced with mCherry filters. Rather than purifying the MBP-pHire protein, equal volumes of bacteria were permeabilized with nigericin and high K+ solution for pH 5 to 9. These aliquots were then used to gather the fluorescence excitation and emission spectra of pHire, allowing us to determine the excitation and emission peaks of pHire as a function of pH. Our results showed peaks at Ex579 nm and Em611 nm, or roughly mCherry. Pelleted bacteria could also be fixed in perfusion chambers, revealing the same calibration curve as in mammalian cells and flies. Next, stable HEK293-pHire cells were selected to assay acid-base transport using NH4Cl or CO2/HCO3− in perfusion experiments. These HEK-pHire cells have less fluorescence than MBP-pHire bacteria but show the same Ex/Em peaks. However, an in-frame ATG 5’ to pHire in a mouse (adding 23 amino acids), removed pHire pH-dependence and decreased bacterial fluorescent output. Together these results indicate that pHire is biophysically similar in cells tested but that unstructured N-terminal additions block pH-responses. Funding: R25-DK101405, R21-DK129897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intelligent LFT (iLFT) was developed by NHS Tayside to enable further investigation of abnormal liver function test results on a single request from primary care. It combines demographics, clinical details and blood tests using an algorithm to generate a probable diagnosis and investigation/management plan. iLFT is indicated where there is suspicion of chronic liver disease, abnormal standard LFT, or abnormal ultrasound from Primary Care. Standard Liver Function tests and platelet count are performed, with patients with abnormal results cascaded to aetiological screen, and fibrosis scoring. Advice is offered on referral or Primary Care management, depending on the output from the algorithm, with patients with likely fibrosis being seen in a Nurse Led iLFT Assessment clinic for Fibroscan, Cardiovascular and Alcohol risk assessment and lifestyle advice. Patients with cirrhosis are commenced on primary prophylaxis for variceal haemorrhage and hepatoma surveillance as appropriate, prior to onward referral to Consultant liver clinic. iLFT was rolled out to pilot GP practices from January 2022, and fully in May 2022. We present our first year’s experience with iLFT, focussing on the impact of iLFT on secondary care triage of patients with abnormal LFTs. At the end of June 2023, we have had 1114 iLFT requests since launch, with some repeats because of platelet clumping or haemolysis. Of these 243 had initial results within iLFT cut-offs and 86 had an isolated raised ALP < 200, requiring no further testing. 785 requests had the full cascade carried out. A lot of work went into setting up laboratory algorithms, additional testing has also been generated. We used ELF to further stratify patients with high Fib-4 or NAFLD fibrosis scores. 52 of 271 ELF scores were <9.8, leading to a 19% reduction in referral. 63 patients have been seen in the Nurse led clinic, and x are still waiting. Of those assessed, 32 have been discharged, 10 followed up by fibroscan. Of 21 referred to Consultant clinic, 18 have Fibroscan >10kPa, 3 likely autoimmune liver disease. 9 new diagnoses of hepatitis C. iLFT can be rolled out in Primary care across other Boards. It allows reassurance of appropriate patients, with early diagnosis of significant liver disease. We have demonstrated collaboration between clinical and laboratory services in our board and NHS Tayside. iLFT can detect Hepatitis C, independent of risk based screening. We have confirmed the usefulness of a sequential approach to fibrosis assessment.

Pancreatic β cell failure in type 2 diabetes mellitus (T2DM) is attributed to perturbations of the β cell's transcriptional landscape resulting in impaired glucose-stimulated insulin secretion. Recent studies identified SLC4A4 (a gene encoding an electrogenic Na+-coupled HCO3- cotransporter and intracellular pH regulator, NBCe1) as one of the misexpressed genes in β cells of patients with T2DM. Thus, in the current study, we set out to test the hypothesis that misexpression of SLC4A4/NBCe1 in T2DM β cells contributes to β cell dysfunction and impaired glucose homeostasis. To address this hypothesis, we first confirmed induction of SLC4A4/NBCe1 expression in β cells of patients with T2DM and demonstrated that its expression was associated with loss of β cell transcriptional identity, intracellular alkalinization, and β cell dysfunction. In addition, we generated a β cell-selective Slc4a4/NBCe1-KO mouse model and found that these mice were protected from diet-induced metabolic stress and β cell dysfunction. Importantly, improved glucose tolerance and enhanced β cell function in Slc4a4/NBCe1-deficient mice were due to augmented mitochondrial function and increased expression of genes regulating β cell identity and function. These results suggest that increased β cell expression of SLC4A4/NBCe1 in T2DM plays a contributory role in promotion of β cell failure and should be considered as a potential therapeutic target.

Anoctamins typically function as Ca2+ activated Cl− channels (CaCC) or as specific, phospholipid scramblases. GWAS of miniature schnauzers (>40% with recurrent calcium oxalate (CaOx) kidney stones) revealed a risk locus including an anoctamin 4 (ANO4) variant. ANO4 in mammals was initially reported as a phosphatidylserine (PS) scramblase. Urinary extracellular vesicles (EV) show decreased ANO4 protein in human stone formers. However, in Drosophila renal structures (Malpighian tubules, MTs), subdued (the ANO4‐homolog), functions as both a CaCC and scramblase. Since subdued also functions to defend against gram‐negative bacteria, it was knocked down to determine whether CaOx crystals were altered by bacterial infection.Subdued was knockdown (KD) in MT principle cells by crossing Uro:Gal4 flies with UAS:subdued‐RNAi flies. F1‐flies were evaluated using CaOx crystallization in dissected MTs as well as after 4d NaOx feeding protocols. To evaluate infection, uropathic E. coli (UPEC):eGFP were fed to flies or added to NaOx solution before an overnight time‐lapse.Interestingly, the canine ANO4 variant has a 0.01 allele variant frequency at a residue conserved in humans, canines and Drosophila. Rapid crystallization experiments show that MTs with subdued‐KD have fewer CaOx crystals than control MTs. However, prolonged NaOx feeding assays showed that subdued‐KD in MTs produces enlarged CaOx crystals. These feeding assays also showed an unexpected phenotype: crystal clustering and matting structures (e.g., bacterial biofilms). These structures have similar patterning to human kidney stones [Sivaguru et al., PMID: 30213974]. Following a four day feeding of NaOx, when UPEC + NaOx is introduced overnight (ex vivo), a three hour bacterial event occurs, causing crystals to more tightly pack. Finally, when UPEC is introduced during a rapid crystallization experiment, the bacteria heavily localize in subdued‐KD MT lumens while control MTs have little eGFP‐fluorescence. Thus, UPEC facilitates CaOx crystal aggregates in subdued‐KD MTs while the nature of this interaction is unknown. In human kidney, ANO4 localizes in thick ascending limb (NKCC2+), while in murine kidney localizes more proximal in the Loop of Henle (UMOD+). As mice are resistant to CaOx stones, these slight location differences may reveal altered TAL function by ANO4.This Drosophila avatar indicates that subdued (ANO4) normally limits CaOx crystal aggregation and limits UPEC growth. As an apparent pathogenic amino acid is conserved in mammals and flies; subdued’s role in fly bacterial infection suggests an additional ANO4 role in human kidney stone formation. These results indicate that examining antibacterial treatments with UPEC infection may allow an antibiotic approach to more efficiently eliminate kidney stones. Future experiments determine what functional aspect of subdued alters CaOx crystallization. Moreover, this simple kidney stone avatar‐the fly – continues to provide new and mechanistic insights to CaOx stone formation.Support or Funding InformationSupport: Oxalosis & Hyperoxaluria Foundation, R25‐DK101405, Mayo FoundationDrosophila MT (Uro:Gal4 x UAS‐subdued‐RNAi) viewed with (A) DIC for CaOx birefringence, (B) fluorescence to visualize UPEC:eGFP and (C) merged to show coincidence of UPEC with CaOx crystals. The red line indicates the length of the CaOx/UPEC mass. This is not observed in control MTs.Figure 1

Dipteran Na+‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na+‐K+‐Cl− cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na+ conductances than controls. Moreover, in flux assays using Li+ as a Na+ tracer, Li+ transport by aeCCC2 was not dependent upon extracellular Cl− and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl−‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na+ replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na+‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na+ with saturable Michaelis‐Menton kinetics. The apparent affinity for Na+ was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li+ and Rb+ were also detectable when added to the bathing saline as tracers for Na+ and K+. In w118‐control flies, fluid secreted by MT measured using these procedures had a [K+] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na+] of 61.2 ± 7.8 mM. In preliminary experiments, [K+] was 112.7 ± 1.3 mM (n=4) and [Na+] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.Support or Funding InformationFunding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.

Dysregulation of glucose homeostasis in Type 2 diabetes mellitus (T2DM) is attributed to pancreatic β‐cell failure in maintaining adequate glucose‐stimulated insulin secretion (GSIS) to compensate for diminished insulin action. Recent single cell transcriptomics studies of human pancreatic islets have identified Slc4a4 (encoding an electrogenic Na+‐nHCO3− cotransporter, NBCe1) as a gene repressed in healthy non‐diabetic β‐cells, but expressed in human T2DM β‐cells. However the role of Slc4a4/NBCe1 in pancreatic β‐cell function remains unknown. Thus, in the current study we set out to test the hypothesis that mis‐expression of Slc4a4 in β‐cells contributes to loss of GSIS and impaired glucose homeostasis in T2DM. Subsequently, we first assessed Slc4a4 mRNA expression in isolated human islets (Control vs. T2DM) by qPCR and quantified NBCe1 protein expression by immunofluorescence in autopsy‐derived human pancreas tissue (Control vs. T2DM). We observed a 4‐fold induction in Slc4a4 mRNA expression in T2DM islets compared to non‐diabetic controls (p=0.004 vs. Control) and significantly increased NBCe1 co‐localization with insulin‐positive β‐cells of T2DM patients compared to controls (p<0.001 vs. Control). In addition, we also demonstrated that chronic inhibition of NBCe1 activity using the channel specific inhibitor (S0859) significantly enhanced GSIS in islets isolated from T2DM patients (~1.5‐fold increase vs. untreated, p=0.006). To delineate the role NBCe1 plays on β‐cell function in vivo, we generated β‐cell specific knockout (β‐Slc4a4−/−) mice by crossing Slc4a4fl/fl (G. Shull, UC) and Ins2‐Cre mouse models. We confirmed >50% reduction in Slc4a4 mRNA expression in β‐Slc4a4−/− islets and complete ablation of NBCe1 protein expression. In order to model the environmental stress that leads to metabolic defects common to T2DM, we exposed β‐Slc4a4−/− mice and control littermates to either standard chow diet or 60% high fat diet (HFD) for 8 weeks ad libitum. Under chow‐fed conditions, β‐Slc4a4−/− mice displayed normal glucose tolerance (p>0.05 vs. β‐Slc4a4+/+ Chow). Interestingly, β‐Slc4a4−/− mice exposed to HFD exhibited enhanced glucose tolerance and GSIS (p<0.05 vs. β‐Slc4a4+/+ HFD) and thus were protected from development of T2DM phenotype characteristics of HFD treatment. Finally, to understand the mechanism by which Slc4a4 deletion enhanced β‐cell function, we performed RNA‐sequencing on islets isolated from β‐Slc4a4−/− and β‐Slc4a4+/+ mice exposed to chow diet or HFD. Notably, gene‐set enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) defined biological pathways revealed that Slc4a4 deletion enriched genes associated with the cell cycle (p<0.001 vs. β‐Slc4a4+/+ Chow; p<0.001 vs. β‐Slc4a4+/+ HFD), while suppressing genes associated with apoptosis (p=0.002 vs. β‐Slc4a4+/+ Chow; p=0.004 vs. β‐Slc4a4+/+ HFD) in the presence of both chow diet and HFD. These result suggest that mis‐expression of Slc4a4 plays a novel role in the induction of β‐cell failure in T2DM. Future studies will delineate exact molecular mechanisms underlying NBCe1‐mediated regulation of β‐cell function in health and under conditions of metabolic stress.Support or Funding InformationMayo Clinic Center of Biomedical DiscoveryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Human mutations in the Na+ bicarbonate cotransporter NBCe1 (Slc4a4) cause proximal renal tubular acidosis (pRTA), cataracts & glaucoma. We reported that whole-gene nbce1 knockouts (KO) are acidotic (blood pH <7.1) and aged, heterozygous mice (HET) have ­↑IOP (intraocular pressure) [IVOS 2013]. One patient (Q29X; only affects NBCe1A, i.e., kidney & eye), has pRTA & glaucoma. We characterized the phenotypes of an NBCe1A isoform-knockout mouse (KOA) and compare to HET & KO phenotypes, & to nbce1A mice (HETA, WTA). Methods: We mated HET mice to generate KO's. Using TALEN-technology, we generated an isoform, KOA mouse [IVOS 2014]. IOPs were measured with a rebound Tonometer. Blood chemistries were measured using a pHOx Ultra analyzer. Kidneys and eyes were fixed, embedded and sectioned for histology. Results: Both KO & KOA are runted. KOA mice live longer than KO mice (>400 d vs 18 d). Both KOA and KO mice are acidotic (7.10±0.07; 7.08±0.04) with low blood [HCO3-] (mM:7.2±1.5; 4.6±1.0). KOA and KO mice have elevated renin, angiotensin II, and aldosterone levels, i.e. RAAS activation. As HETs age (>1y), their IOP's ­↑(19.8 mmHg) while WT IOPs (15.2 mmHg) do not. KOA mice show ­↑IOP at ~1y (17.6 mmHg) compared to HETA & WTA (15.2, 12.7 mmHg). Renal histology of KO & KOA show cortical cysts. Conclusions KOA mice survive longer (>1y), are acidotic, have ↑­IOP and cysts. The onset age of ­↑IOP is later and less extreme than IOP & pH of whole-gene nbce1 KO. Thus, NBCe1A is the major renal HCO3- absorption path, is RAAS controlled, controls ocular fluid transport (ΔIOP) and causes cysts. These mice should facilitate both aging and systemic disease studies. Support: DK101405; Kogod Aging Center